Cholera toxin, the secretory product of Vibrio cholerae responsible in part for the devastating diarrheal syndrome characteristic of cholera, activates adenylyl cyclase by catalyzing the ADP-ribosylation of Gs(alpha), the stimulatory guanine nucleotide-binding protein of the cyclase system. This toxin-catalyzed reaction is stimulated, in the presence of GTP, by about 20 kDa guanine nucleotidebinding proteins, termed ADP-ribosylation factors or ARFs. Two forms of ARF, sARF I and sARF II, were isolated from bovine brain cytosol. Rabbit polyclonal antibodies against bovine sARF II reacted with soluble and membrane ARFs but did not react with other guanine nucleotide-binding proteins such as the 20 kDa protein ras and the heterotrimeric transducing G proteins (e.g., Gt(alpha), Gs(alpha), Gi(alpha), and Go(alpha)). The anti-ARF antibodies recognized about 20 kDa ARF-like proteins in a variety of species and organ systems. The highest levels of immunoreactivity were observed in brain and other neural tissues. In these tissues an ARF doublet was observed, with the upper band (sARF II) being the predominant form. In other tissues, an immunoreactive band corresponding to the lower molecular weight species (sARF I) was present at higher concentration. Levels of ARF, when quantified by immunoreactivity, correlated with those determined by a functional assay, stimulation of cholera toxin-catalyzed ADP-ribosylation. During rat brain development, when quantified by both immunoreactivity and function, sARF II was lowest at birth, showed some increase at 10 days, and was maximal at 27-60 days; sARF I was unchanged. In spleen and heart, sARF I predominated over sARF II; in spleen, it increased with age while in heart, it decreased. Based on these studies, it appears that ARF proteins share epitopes and are expressed at different levels during development.